Most of the interior of a cell is water, and life could not exist without circulating water: Nutrients and ions that are vital for our existence are solvated by water, and waste products are removed by circulating currents. Therefore, all three kingdoms of life, from the simplest unicellular organisms to humans, express specialized membrane-embedded proteins that form pores for water conduction called aquaporins. Because of their abundance and their role in maintaining celluar homeostasis, many diseases and cancers are directly related to misregulation or mutation in aquaporins. We study aquaporin 0 (AQPO), a water channel from the eye lens, causing severe lesions in the lens, cataracts and blindness when mutated. AQPO tightly regulates water permeability within the eye lens by responding to changes in pH, as well as by Ca2*/calmodulin. Calmodulin (CaM) is a ubiquitous cytosolic protein commonly coupled with Ca2+ regulation. It is proposed that these two AQPO regulatory signals are separable, and that AQPO exists in two functional states: a low water permeability resting state at pH 7.2;and a high permeability state induced by a drop in pH or mediated by Ca2VCaM. Moreover, we suggest that these regulatory signals lead to major conformational changes in AQPO, causing water channels to open/close and permeability to increase/decrease. The aims of this proposal are to use cryo electron microscopy (cryo EM) and electrophysiology to unravel these two regulatory mechanisms by studying the conformational changes in AQPO as its water channels open/close in response to pH and Ca2+/CaM. Results from this study have wide implications in the fields of water channel regulation, physiology as well as broader implications to the Ca2VCaM signaling system and to membrane and cellular biology in general. Success in achieving the aims set in this proposal would additionally mark a technological achievement in the field of structural biology by providing the first structure of a membrane protein embedded in a membrane and bound to a soluble protein. It is hoped that by gaining a fundamental understanding of how cellular water homeostasis is regulated by aquaporins, we may begin to better understand the disease states and cancers associated with their misregulation.